OBJECTIVE To determine numbers of SIV-specific CTL in SIV-infected rhesus macaques. RESULTS A tetrameric recombinant major histocompatibility complex (MHC) class I-peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and 2-microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C-M) representing the optimal nine-amino acid peptide of Mamu-A*01-restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C-M complex. Tetrameric Mamu-A*01/p11C, C-M complex bound to T cells of SIVmac-infected, Mamu-A*01+, but not uninfected, Mamu-A*01+, or infected, Mamu-A*01 rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01+ rhesus monkeys was only found in the cluster of differentiation (CD)8/+ T lymphocyte subse t and the percentage of CD8/+ T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01+ rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C-M complex-binding, but not nonbinding, CD8/+ T cells. Furthermore, the percentage of CD8/+ T cells binding this tetrameric Mamu-A*01/p11C, C-M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. DISCUSSION These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings. FUTURE DIRECTIONS This reagent will be useful for assessing vaccine efficacy. KEY WORDS MHC, CTL, tetramer, rhesus macaque, SIV FUNDING NIH RR00167, AI32426, AI41913 PUBLICATIONS Kuroda, M.C., Schmitz, J.E., Barouch, D.H., Craiu, A., Allen, T.M., Sette, A., D.I. Watkins, Forman, M.A., and Letvin, N.L. 1998. Analysis of gag-specific cytotoxic T lymphocytes in SIVmac-infected rhesus monkeys by cell staining with a tetrameric MHC Class I/peptide complex. J. Exp. Med. 187:1373-1381.